The innate immune response is a complex and highly regulated process. The goal of this system is to provide a broad spectrum and rapid host protection from invading pathogens, as well as to facilitate the activation and further development of the acquired immune response. In recent years, a large body of evidence has indicated that the family of Toll-like receptors (TLRs) plays a critical role in the activation of the innate and the inflammatory response (Foster et al., 2009; Mitchell et al., 2007; O'Neill et al., 2003).
TLRs are a family of conserved transmembrane receptors composed of an extracellular, a single transmembrane, and a cytoplasmic Toll-interleukin1 receptor-resistance (TIR) domain. These receptors recognize a wide variety of ligands, named pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) from gram-negative bacteria, lipoteichoic acid (LTA) from gram-positive bacteria and flagellin, as well as many other compounds. TLRs also help the innate immune system to identify necrotic events, which result in the clearance of various “contaminations” from the surroundings (damage associated molecular pattern molecules, DAMPs). Many tumor cells undergo necrosis mediated by the immune system and may lead to further activation of an inflammation response via TLRs.
Upon binding to their specific PAMP these receptors can either form homodimers, such as in the case of TLR4, or heterodimers as in the case of TLR2 with TLR1 and TLR6. For example, LTA causes a hetero-dimerization of TLR2 with TLRs 1 or 6, and LPS (as a complex with the protein MD2), causes TLR4 homo-dimerization (Ozinsky et al., 2000; Hajjar et al., 2001; Lee et al., 2003; Jin et al., 2007). This triggers a down-stream signaling cascade, resulting in the activation of the NFκB and other survival pathways such as P38 and JNK. One of the main products of TLR activation is the secretion of TNFα, a major proinflammatory cytokine that has many roles in the progression of the inflammatory process. TLR over-expression or over-activation is associated with a variety of diseases e.g. systemic inflammatory response syndrome (SIRS), organ failure, exacerbation of latent or active viral infections (e.g., infection with HIV, cytomegaloviruses, herpes simplex, and influenza virus), inborn or acquired predisposition to pulmonary bacterial infection, congestive heart failure with pulmonary edema, chronic obstructive pulmonary disease, multiple myeloma, SLE, lupus, ulcerative colitis, Crohn's disease, autoimmune diseases such as multiple sclerosis, rheumatoid diseases, chronic hepatitis, malaria (P. Falciparum), neuritis, viral encephalitis (West Nile), candidiasis, atherosclerosis, degenerative diseases, neurodegenerative diseases, and various types of cancer. In extreme cases, severe unregulated activation of the inflammatory process might lead to septic shock, organ failure and death (Foster et al., 2009; Meng et al., 2004).
Attempts to regulate over expression or over activation of TLRs include mainly the use of TLR antagonists. US 2010/062026 discloses agents with poly TLR antagonistic activity (mycobacterium) useful in management of diseases wherein TLRs are over expressed. WO 2009/019260 discloses a method for reducing the biological activities of TLR2 in ischemia reperfusion injury, using TLR2 antagonists. WO 2009/047791 discloses novel synthetic TLR antagonist, potentially useful in the treatment of inflammation, autoimmunity, allergy, asthma, graft rejection, graft versus host disease, infection, sepsis, cancer and immunodeficiency. U.S. Pat. No. 7,410,975 discloses a method for modulating signaling through TLRs using small molecule TLR antagonists. US 2006/0211752 discloses a method of treating a TLR-mediated disease or disorder using methimazole (thyroid hormone antagonist) derivatives and/or cyclic thione derivatives.
Inflammation has been linked to cancer formation and progression and has been studied extensively during the past decade. In colon cancer, inflammation caused by H. pylori has been shown to lead to the formation of many mutations in colon cells including the expression of oncogenes and down regulation of tumor suppressor genes. Other cancers include: hepatocellular carcinoma (HCC) via inflammation induced by hepatitis B virus (HBV), gastric cancer, and lung cancer.
Indeed, TLRs have been demonstrated to be expressed and activated in various solid tumors but mostly not on normal cells in these organs. In addition, there are differences in the array of TLRs identified in different types of tumors (Sato et al., 2009). Overall, these findings suggest that the tumor has a selective mechanism by which it “decides” what type of TLR array will be expressed. The expression and activation of the various TLRs modulate the microenvironment of the tumor and protect it from clearance by the immune system.
To date most studies on prostate cancer were focused on TLR4 and TLR9 which are related to the two main types of infections, bacterial and viral, identified in prostate samples. TLR4 recognizes components from gram-negative bacteria and TLR9 recognizes single stranded DNA from viruses (Huang et al., 2005). Importantly, down regulation of the expression of these proteins leads to a decrease in prostate tumor growth, metastasis formation and mortality in mice (Kundu et al., 2008). However, these studies used siRNA technology which, although highly effective in-vitro, is difficult to apply in-vivo.
WO 03/045431 discloses methods for treating cancer using a combination of a tumor-derived dendritic cell inhibitory factor antagonist, namely a IL-10 or IL-10R antibody, and a TLR9 agonist.
Recent studies demonstrated a significant number of incidences of infection by gram-positive bacteria in prostate sections derived from prostatectomy patients at various stages of the disease (Saenz-Abad et al., 2008; Sfanos et al., 2008). Therefore we hypothesize that TLRs 1, 2, and 6 that are activated by lipoteichoic acid also play a role in prostate cancer.
As stated above, unregulated TLR activation may lead to neurodegenerative diseases such as Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by neuronal loss, activation of microglia, reactivate astrocytes and accumulation of intracellular and extracellular aggregates. According to the “Amyloid hypothesis”, extensive extracellular deposits of fibrillar β amyloid (fAβ) condensed to form senile plaques in the brain which are the leading cause of neurodegeneration in AD. It posits that imbalance in the production and clearance of Aβ leads to an increase in its steady-state levels within the brain over the course of decades, resulting in the complex molecular and cellular changes within the brain that characterize Alzheimer's disease. Aβ is produced by the cleavage of APP (amyloid precursor peptide) by either α or β secretase and subsequently the γ secretase resulting in two major forms being 40 and 42 residues in length. In a normal individual, the majority of the Aβ produced is the 40 amino acid species, whereas 5-15% of the total Aβ forms are Aβ42. However, Aβ42 is more toxic and form more stable fibrils and is thus more related to Alzheimer's disease.
Extensive research has demonstrated the involvement of inflammation in Alzheimer's disease. The principal immune effector cells of the brain are microglia cells that are known to be recruited to sites of fAβ plaques in Alzheimer's disease. These cells showed an activated phenotype and high levels of proliferation when surrounded with fAβ, suggesting the role of those cells in the inflammatory phenotype characterizing Alzheimer's disease. Removal of microglia cells from culture containing mixed brain cell and fAβ almost totally eliminates the toxic effects of fAβ on primary neurons, implying that microglia or their products mediate the neurotoxic effects of fAβ. Although microglia and macrophage cells show a role in the progression of Alzheimer's disease, some aspects of the microglial inflammatory response represent positive influences with respect to Alzheimer's disease pathogenesis, such as phagocytic clearance of Aβ from the brain. However, prolonged damage from microglia-mediated inflammatory response likely exacerbates disease pathogenesis. Moreover, the levels of proinflammatory cytokines depend on the magnitude of plaque existence in the Alzheimer's disease brain.
Some TLRs employ additional co-receptors that assist in pathogen recognition, such as CD14 for TLR4. Recently it was demonstrated that TLR4 mediates extensive neuronal cell death upon LPS treatment in microglia cell culture and more importantly in vivo (Lehnardt et al., 2003). This finding suggesting that TLR4, a receptor so far associated with defense against microorganisms may be relevant in chronic neuroinflammation in Alzheimer's disease. Only recently have TLRs been implicated in microglial activation in Alzheimer's disease and how TLRs function in the inflammatory response in AD is now under active investigation. Using a variety of techniques, CD14 was shown to bind fAβ42. It was further shown that the interaction between fAβ42 and CD14 is 20 times greater than that between CD14 and non fAβ, indicating the importance of the fibrillar structure of Aβ in binding CD 14. TLRs have been shown to have a role in neuronal apoptosis as well. Hippocampal neurons exposed to conditioned media from microglia treated with fAβ resulted in neuronal death. However, media from CD14−/− or TLR4−/− microglia cells treated with fAβ were unable to kill neurons (Fassbender et al., 2004 and Walter et al., 2007). These data suggest that CD14 and TLR4 function in the production of neurotoxic molecules. Moreover, it was recently found that neurons express several TLRs and that TLR4 expression increase in neurons when exposed to fAβ, leading to neuronal apoptosis. This finding suggests that neurons expressing TLR4 are vulnerable to degeneration in AD (Sung-Chun et al., 2008).